WO2018170624A1 - Method for reducing mir-185 and mir-424 expressions - Google Patents

Abstract

Provided is a method for reducing miR-185 and miR-424 expressions. The method comprises constructing a eukaryotic expression vector pRI-185-424 targeting to miR-185 and miR-424 over a eukaryotic vector pRI-GFP/Neo that contains an H1 promoter, thereby down-regulating miR-185 and miR-424 expressions.

Description

 Description Title: Method for reducing expression of microRNA-185 and microRNA-424

 [0001] The present invention relates to the field of life sciences, and more particularly to a method of reducing the expression of microRNA-185 and microRNA-424.

 Background technique

 [0002] MicroRNA-185 (miR-185) is a 22 nt miRNA located in human chromosome 22ql l.

 21, plays an important role as a tumor suppressor gene in the development and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, etc. In addition, it is a methylation-related tumor suppressor miRNA that can be directly targeted. The expression of DNMT1 affects the methylation level of the whole genome, which in turn regulates the methylation status of certain genes, affecting gene expression; microRNA

 -424 (miR-424) is a miRNA discovered in recent years, which affects the biological effects and development of tumor cells by acting on target genes in various tumors, thereby affecting the biological effects and development of tumor cells. The role of genes, tumor suppressor genes, or promote, inhibit tumor invasion and metastasis. Studies have shown that miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-α; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing. By controlling the expression of miR-185 and miR-424, peers can work with other drugs to provide new epigenetic ideas for the treatment of cancer.

 technical problem

 [0003] At present, miRNA function research is generally achieved by miRNA overexpression and silencing. miRNA silencing is the presentation of artificially synthesized oligonucleotide small molecules into cells, and endogenous miRNAs.

 The formation of a heteroduplex allows the miRNA to reduce the inhibition of the target gene to achieve regulation of gene function. Silencing of miRNAs, especially long-term stable silencing, is currently difficult to achieve. Commonly used methods for silencing miRNA mainly include anti-miR, antagomiR, miRNA sponge, etc. These methods have short effects such as short inter-turn, poor stability, complex construction, high requirements on instrument conditions, and complicated technical operations, which are difficult to achieve, simple, and effective. The effect of miR-185 and miR-424 expression was reduced.

Problem solution Technical solution

 [0004] The object of the present invention is to provide a method for reducing the expression of miR-185 and miR-424, which is simple in design, easy to operate, and can easily and effectively down-regulate the expression of miR-185 and miR-424, Overcoming the deficiencies of the prior art.

 [0005] A method for the present invention: a method for down-regulating expression of miR-185 and miR-424, and constructing eukaryotic expression targeting miR-185 and miR-424 on a eukaryotic vector pRI-GFP/Neo containing a HI promoter Vector pRI-185-424, which regulates the expression of miR-185 and miR-424 in eukaryotic cells; after transfecting the vector into eukaryotic cells in vitro, the expression of miR-185 and miR-424 was detected by real-time PCR Level.

 [0006] The construction of the pRI-185-424, specifically, the sequence design of the Tud-185-424 is completed by software.

The sequences of pRI-GFP/Neo and Tud-185-424 were double-digested with Xho I and Bgl II, and the recovered fragments were purified and ligated overnight at 4 ° C. After ligation, single colonies were picked and inoculated. In LB medium containing l (Vg/ml kanamycin, 12 hours later, the bacterial solution was extracted and sent to Shanghai Yingjun for sequencing. The correctly sequenced vector was the successfully constructed recombinant plasmid pRI-185-424.

 Advantageous effects of the invention

 Beneficial effect

 [0007] The present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-185-424 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-185 and miR-424; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-185 and miR-424 was detected by a real-time PCR instrument. The invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

 Brief description of the drawing

 DRAWINGS

 1 is a schematic diagram showing the structure of the pRI-GFP/Neo vector; FIG. 2 is a quantitative PCR method for detecting the expression level of miRNA, wherein a. miR-185 expression, b. miR-424 expression.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

The present invention can be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the invention. The invention as described in detail in the claims should not be limited or limited.

Example of down-regulating expression of miR-185 and miR-424

1 [0011] 1

 First, the sequences of hsa-miR-185 and hsa-miR-424 were searched in the miRBase database, and the Tud RNA targeting hsa-miR-185 and hsa-miR-424 was designed according to the design principle of Tough Decoy RNA (Tud RNA). Finally, add Xho I and Bgl to the 5' and 3' ends of the Tud RNA.

 The enzyme was digested to obtain the desired Tud-185-424 sequence. The nucleotide sequence of Tud-185-424 is shown as SE Q ID No 1.

[0012] 2 using Xho l and 8 _§ 1 11 double-cleavage of the backbone vector 1-0?? 60 and 1 1 (1-185-424 sequence, purification of the recovered fragment, overnight at 4 ° C; after the connection After transformation, 6 colonies were picked and inoculated into LB medium containing 10 g/ml kanamycin. After 12 hours, the bacterial solution was extracted and sent to Shanghai Yingjun for sequencing. The correct vector was the successfully constructed recombinant plasmid. pRI-185-424.

 [0013] 3 The endotoxin-free plasmid extraction kit (Tiangen Biochemical) was used to extract the recombinant plasmid pRI-185-424.

 [0014] 16HBE cells were seeded in a 6-well plate at a density of 30,000/ml, and 10

 Plasmid pRI-185-424 was transfected with Lipofectamine 2000 and continuously cultured at 5% CO 2 at 37 ° C. After 48 sputum, total RNA was extracted from each group, using Realtime.

 The expression of miR-185 and miR-424 was detected by PCR, and the results showed that the expression levels of m iR-185 and miR-424 were significantly down-regulated in the pRI-185-424 transfection group (Fig. 2), suggesting that the vector can be used for The expression of miR-185 and miR-424 was reduced.

 Industrial applicability

 [0015] The present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-185-424 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-185 and miR-424; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-185 and miR-424 was detected by a real-time PCR instrument. The invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

Claims

Claim  [Claim 1] A method for reducing microRNA-185 and microRNA-424, characterized by:  The eukaryotic expression vector pRI-185-424 targeting miR-185 and miR-424 was constructed on the eukaryotic vector pRI-GFP/Neo of the promoter, and the expression of miR-185 and miR-424 in eukaryotic cells was regulated below. After the vector was transfected into eukaryotic cells in vitro, the expression levels of miR-185 and miR-424 were detected by a real-time PCR instrument.  [Claim 2] Method for reducing expression of microRNA-185 and microRNA-424 according to claim 1 , characterized in that: the construction of the pRI-185-424, specifically, the sequence design of Tud-185-424 is completed by software; the backbone vectors pRI-GFP/Neo and Tud are double-digested with Xho I and Bgl II - 185-424 sequence, purification of the recovered fragment, and overnight ligation at 4 °C; transformation after ligation, picking a single colony, inoculation into LB medium containing 1 (Vg/ml kanamycin, 12 hours) The bacterial liquid was extracted and sent to Shanghai Yingjun for sequencing. The correct vector was the successfully constructed recombinant plasmid pRI-185-424.